Portable single unit device for ochratoxin a (ota) toxicity analysis for rice quality monitoring

ABSTRACT

A single unit, handheld field portable apparatus and method for analyzing Ochratoxin A (OTA) in rice quality monitoring, based on fluorescence signal output. Aliquots may be analyzed by adding at least one or more reagents to the sample aliquot that reacts selectively with an analyte contained therein. The reaction product, which is selective for the analyte of interest and proportional to its concentration, is measured with an appropriate detector. This enables simple and accurate testing of samples using time honored wet-chemical analysis method in microliter volume regimes while producing remarkably small volumes of waste. The device includes a multipurpose controller board for processing and analysis purpose, a camera which is integrated with the controller, a resistive touch liquid crystal display to view the results, a light emitting diode to emit the UV light, and a power bank. The device may operate using a touch display.

FIELD OF THE INVENTION

The present invention relates to defining the levels of OTA in ricequality monitoring and more particularly to a new, inexpensive,portable, hand held means for testing in a field environment for thepresence of OTA, using fluorescence output signal.

DISCUSSION ON THE BACKGROUND

The presence of unsafe levels of chemical compounds, toxins andpathogens in food represents a serious threat to the safety of foodsupply and public health, especially in countries with a poor economy.Among chemical hazards causing foodborne dysfunctions, mycotoxinsparticularly OTA, pose particular challenges due to their extremely hightoxicity at low exposure levels. In spite of significant progress,available methods are expensive; require sample collection and transportat a centralized lab, skilled personnel and specialized equipment foranalysis.

Optical spectroscopy finds potential applications in diagnostics,pharmacological testing, food quality assessment, and environmentalsensing among numerous other applications. It is convenient forqualitative and quantitative analysis due to its qualities of being easyto use and non-destructive. Spectrometers commonly used in laboratory orindustry for sample testing are expensive, large size and requirecomputer setup detector to analyze the information received,consequently spectroscopic testing is restricted to controlledenvironment laboratory. Recently, a lot of work has been done togenerate portable spectrometers, though they also require externalcomputing system for collection and analysis of data. These qualitiesremarkably raise the price of the system and restrict the system'srange. These restrictions lead to approach readily accessibleinterfacing devices with spectrometer. Mini quantum dot and mobile phonebased spectrometers have been reported, however no single hand-heldportable device has been reported.

SUMMARY

Machine vision is an automated process that integrates many processesfor visual perception, such as image acquisition, image processing,classification, recognition, and decision. Machine vision includestechniques to estimate the characteristics of objects in the image, tomeasure object geometry, and to interpret information geometry. Machinevision process comprises three main activities such as image capturing,image processing, and image analyzing. In this research, the machinevision system is embedded in a single hand-held portable device. Weexhibit first of its type standalone, economical, compactly designed,accurate and unique handheld portable device based on fluorescencesignal. The device does not require any desktop computers or laptopswith heavy software installation neither Wi-Fi nor internet to work. Itis self-assembled minicomputer with multipurpose controller board, acamera which is integrated with controller, a resistive touch crystaldisplay for monitoring results analysis, a light emitting diode to emitthe UV light, and a power bank such as a portable power bank. Analysisis performed within a few seconds and we can get the result on thedisplay in term of numbers and percentages. It does not require datainterpretation or an expert to operate it. We present a field portabledevice which is easy to operate and suitable for decentralized and onsite testing. We have utilized the device to monitor OTA as a type ofmycotoxins for rice quality monitoring.

A single unit, handheld field portable apparatus (1.8) and method foron-site analysis of OTA in rice quality control, using fluorescenceoutput signal (008) is reported. The invention enables simple andaccurate testing of samples using time honored wet-chemical analysismethods in microliter volume regimes while producing remarkably smallvolumes of waste. Two types of nanoprobes including nanoceria (ceriumoxide nanoparticles) and nitrogen doped titania (N-doped titanium oxidenanoparticles) are reported to amplify the output generated signal(008). The device includes a multipurpose controller board (1.1) forprocessing and analysis purpose, a camera (1.3) which is integrated withthe controller (1.1), a resistive touch liquid crystal display (1.7) toview the results (008), a light emitting diode to emit (1.5) the UVlight (003), and a power bank (1.2) such as a portable power bank. Thedevice may operate with a single click using touch display.

Only a limited number of samples can be tested and predictive or earlywarning information is missing. This is a significant impediment toprovide timely counter-measures, especially in remote locations and incountries with limited resources. The present invention may address thisneed by providing an apparatus and method suitable for on-site analysis.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the invention will become apparent fromthe following description of some forms of embodiment of the invention,given as a nonlimiting example, with the help of the appended diagramsillustrated in the attached drawings, in which:

FIG. 1 represents the actual design of prototype and schematicpresentation of the hardware components used in the fabrication of theprototype.

FIG. 2 is a flow chart illustrating the programming and functions togenerate output signal and subsequently the display for users.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

The device includes a multipurpose controller board (1.1) for processingand analysis purpose, a camera (1.3) which is integrated with thecontroller, a resistive touch liquid crystal display (1.7) to view theresults (008), a Light emitting diode (1.5) (wavelength in the range of350 nm-500 nm depending on the toxin of interest) to emit the UV light,Micro secure digital (SD) card (1.6) and a portable power bank (1.2). Aplastic cuvette (1.4) can be used as a sample holder.

The device operates (001) using a touch display (1.7) to boot the system(002). The camera (1.3) captures an image in a black box with UVexcitation (003) and sends the image (004) to the brain of the devicewhich is controller board (1.1). The image (004) is processed (e.g., ina JPG file). The noise of the image is filtered (005) by using specialalgorithms (007). Finally, to determine the level of OTA in rice sample,several steps are as follows, i.e. determination of the average pixelvalue of the image resulted from image segmentation using imageprocessing algorithm (007), conversion of the original image (004), aswell as assignment of value. The controller board is programmed in aunique and simple way to extract details (006) of the image (004) usingimage processing algorithms (007). Based on the programmed algorithm(007), the smart controller (1.1) will analyse the pixel by pixelinformation of the image (004), which is processed by the controller(1.1), and make a decision on the basis of machine learning algorithms.The process is to determine the center point of identified object todetermine the object area. The area is then analyzed to measure theaverage and deviation standard of pixel values.

EXAMPLE Experimental Details

A stock standard solution of 1 mg/ml was prepared by dissolving 5 mg ofOTA in 5 ml of methanol and then stored at −20° C. OTA solutions inmethanol stored at −20° C. are stable over an extended period of time.Working standard solutions in the concentration level of 0.5-200 ng/mLwere prepared by diluting the stock solution. pH was adjusted to 7.4. 2mL of the working standard solutions were added in the plastic cuvettes(1.4) to perform the testing in the prototype (1.8). The prototype (1.8)generated images and RGB were used to build the library.

Use of nano probes in the sensing of analytes has been increasedtremendously. Nanomaterial based signal amplification have gained muchattention with additional benefits for rapid analysis. Cerium oxidenanoparticles were used to amplify the fluorescence based signal of thetested analyte. A stock standard solution of 1 mg/ml was prepared bydissolving 5 mg OTA in 5 ml of methanol and then stored at −20° C. OTAsolutions in methanol stored at −20° C. are stable over an extendedperiod of time. Working standard solution at the concentration level of5 ng/mL was prepared by diluting the stock solution. pH was adjusted to7.4. 2 mL of the working standard solutions and nanoceria particle atconcentration of 50 ng/mL were added in the plastic cuvettes (1.4) toperform the testing in the prototype (1.8). The prototype (1.8)generated images and RGB were used to build the library.

Acetonitrile was employed as the testing medium. The signalamplification efficiency of two nanoprobes cerium oxide and N-dopedtitanium oxide nanoparticles was monitored.

-   -   a) A stock standard solution of 1 mg/ml was prepared by        dissolving 5 mg OTA in 5 ml of methanol and then stored at        −20° C. OTA solutions in methanol stored at −20° C. are stable        over an extended period of time. Working standard solution at        the concentration level of 5 ng/mL was prepared by diluting the        stock solution in the solvent acetonitrile-water (6:4, v/v). 2        mL of the working standard solutions in the solvent        acetonitrile-water (6:4, v/v), and nanoceria particle at        concentration of 50 ng/mL were added in the plastic cuvettes        (1.4) to perform the testing in the prototype (1.8). The        prototype (1.8) generated images and RGB were used to build the        library.    -   b) A stock standard solution of 1 mg/ml was prepared by        dissolving 5 mg OTA in 5 ml of methanol and then stored at        −20° C. OTA solutions in methanol stored at −20° C. are stable        over an extended period of time. Working standard solution at        the concentration level of 5 ng/mL was prepared by diluting the        stock solution in the solvent acetonitrile-water (6:4, v/v). 2        mL of the working standard solutions in the solvent        acetonitrile-water (6:4, v/v), and N-doped Titanium oxide        particle at concentration of 50 ng/mL were added in the plastic        cuvettes (1.4) to perform the testing in the prototype (1.8).        The prototype (1.8) generated images and RGB were used to build        the library.

Food authorities all over the world have established a permissible limitof 3 μg/Kg of OTA in cereal samples. Therefore, OTA at a level of 3μg/Kg was used to build the library. Certified Rice samples were used toconstruct the library for future field applications.

-   -   a) Sample of rice is finely ground using mortar and pestle and        was spiked with OTA at concentration of 3 μg/kg. The weighed        crushed rice (2 g) were extracted in 4 mL of solvent mixture of        acetonitrile-water (6:4, v/v) in glass vials. Extraction was        carried out for 10 min using manual shaking till the clear        solvent changes its color to milky solution. Extract was        filtered using whattman filter paper (cat. no. 1001 12.5). 2 mL        of the filtrate solution were added in the plastic cuvettes        (1.4) to perform the testing in the prototype (1.8). The        prototype (1.8) generated images and RGB were used to build the        library.    -   b) Sample of rice was finely ground using mortar and pestle and        was spiked with OTA at concentration of 3 μg/kg. The weighed        crushed rice (2 g) were extracted in 4 mL of solvent mixture of        acetonitrile-water (6:4, v/v) in glass vials. Extraction was        carried out for 10 min using manual shaking till the clear        solvent changes its color to milky solution. Extract was        filtered using whattman filter paper (cat. no. 1001 12.5). 2 mL        of the filtrate solution and nanoceria particle at concentration        of 50 ng/mL were added in the plastic cuvettes (1.4) to perform        the testing in the prototype (1.8). The prototype (1.8)        generated images and RGB were used to build the library.    -   c) Sample of rice was finely ground using mortar and pestle and        was spiked with OTA at concentration of 3 μg/kg. The weighed        crushed rice (2 g) were extracted in 4 mL of solvent mixture of        acetonitrile-water (6:4, v/v) in glass vials. Extraction was        carried out for 10 min using manual shaking till the clear        solvent changes its color to milky solution. Extract was        filtered using whattman filter paper (cat. no. 1001 12.5). 2 mL        of the filtrate solution and N-doped Titanium oxide at        concentration of 50 ng/mL were added in the plastic cuvettes        (1.4) to perform the testing in the prototype (1.8). The        prototype (1.8) generated images and RGB were used to build the        library.

We claim:
 1. A device comprising: a display interface; a controllerboard, connected to the display interface, configured for dataprocessing and analysis; a camera which is integrated with thecontroller; a camera board to support the camera; a sample holderconfigured to mount a sample; a light emitting diode inserted in a wallof the sample holder and configured so that the sample is in front ofthe light emitting diode; a reagent holder configured to hold extractionreagents and a nano probe; and a power bank that powers components ofthe device.
 2. The device of claim 1, wherein the display is a resistivetouch liquid crystal display.
 3. The device of claim 1, wherein thedisplay is attached with the controller board via Display SerialInterface (DSI).
 4. The device of claim 1, wherein the controller boardis a single board programmable computer (SBC).
 5. The device of claim 1,wherein the camera is attached about 2.5 cm to 3 cm above the sampleholder and a lens/sensor of the camera board is aligned to the center ofthe sample holder.
 6. The device of claim 4, wherein the camera takespower through Camera Serial Interface (CSI) from the SBC.
 7. The deviceof claim 1, wherein the light emitting diode has excitation wavelengthbetween 350-370 nm to excite the analyte and generate a fluorescenceimage.
 8. The device of claim 1, wherein the samples are excited throughUV light.
 9. The device of claim 1, wherein the sample holder holds acuvette of 2.5 mL.
 10. The device of claim 1, for use in on-site sampleanalysis.
 11. The device of claim 1, wherein the sample comprisesOchratoxin A (OTA).
 12. A method of testing Ochratoxin A (OTA),comprising: a) performing extraction on a sample comprising OTA with asolvent; b) inserting the sample into a sample holder with aid of asample cuvette; c) adding detection reagent to the sample; d) excitingthe sample; e) performing image capturing and processing; and f)displaying results on an interface of a device.
 13. The method of claim12, wherein the solvent is a mixture of acetonitrile-water.